WO2024074119A1 - Recombinant collagen and use thereof in cartilage repair matrix - Google Patents

Abstract

A recombinant collagen and the use thereof in cartilage repair matrix. The recombinant collagen comprises a sequence as shown in SEQ ID No.1; the amino acid sequence of the recombinant collagen comprises N basic repeating units, and the basic repeating units contain N1 characteristic amino acid sequences as follows: "G-Xaa1-Xaa2-G-E-Xaa3"; and the 3' ends and the 5' ends of the basic repeating units are linked to form the above-mentioned characteristic amino acid sequence. The recombinant collagen has obvious integrin binding activity, has the effects of promoting cell adhesion, proliferation, differentiation, repairing defects of the cartilage tissue, etc., and has good application prospects.

Description

Recombinant collagen and its use in cartilage repair matrix

This application claims the benefit of (1) Application No. 2022112245930 filed on October 8, 2022, and (2) Application No. 2022113785160 filed on November 4, 2022. The entire contents of the prior applications are incorporated herein by reference.

Technical Field

The invention belongs to the field of biomaterials, and in particular relates to a recombinant collagen and an application thereof in a cartilage repair matrix.

Background technique

Articular cartilage injury has become a common orthopedic disease. Trauma, arthritis and sports-related injuries can all cause articular cartilage injury or defect. The repair of articular cartilage mainly depends on the proliferation and differentiation of chondrocytes to produce sufficient extracellular matrix to repair cartilage defects. However, healthy adult chondrocytes are usually static and highly differentiated cells. Because of their low degree of vascularization, their nutrition mainly comes from synovial fluid and subchondral bone. If traumatic or pathological damage occurs, the repair and regeneration of articular cartilage is very limited. At present, some treatment methods used in clinical practice include microfracture, osteochondral transplantation, chondrocyte transplantation, etc. These methods can repair cartilage defects to a certain extent and relieve patients' pain, but the long-term effect is not satisfactory. The important reason is that the cartilage reconstructed by these methods is very different from normal hyaline cartilage in organization and structure, which leads to a gap in histological and biomechanical properties with normal human cartilage.

In recent years, tissue engineering has made considerable progress in obtaining functional articular cartilage. Some bioengineering treatment strategies, including stem cell methods and scaffold technology, have been proposed to provide minimally invasive and more effective ways to repair articular cartilage. As one of the three major elements of tissue engineering, scaffold materials play an important role in the construction of tissue engineering cartilage. Their mechanical properties and chemical composition will affect cell adhesion, proliferation and cell phenotype. Collagen has become a biomaterial with extensive research in tissue engineering due to its excellent biocompatibility, bioactivity and biodegradability. However, most traditional collagen comes from animals. The amino acid sequence of animal collagen is highly similar to that of human collagen. For example, the similarity between mammalian pigs and cattle and humans is 95%, and the similarity between fish and humans is 65%. However, when animal collagen is used in biomaterials or medical devices, these differences may bring obvious risks such as immune rejection, sensitization, and viral infection and transmission. At the same time, since different subtypes of natural collagen are unevenly distributed in tissues and organs and coexist in a complex form, the efficiency of extracting collagen from biological tissues depends largely on its natural abundance, and it is also difficult to avoid the presence of a mixture of different subtypes. Therefore, the deficiencies in batch stability, purity, molecular weight and distribution of animal-derived collagen further limit its many applications in the biomedical field.

In order to solve the above problems, researchers have focused on the research of using genetic engineering technology to prepare recombinant collagen. Recombinant collagen is based on the natural human collagen gene sequence, and codons with specific functional expressions are selected. After appropriate modification and editing, they are transcribed into host cells and the recombinant protein material is obtained by large-scale production using biofermentation technology.

Summary of the invention

The technical problem to be solved by the present invention is to provide a recombinant collagen and its use in a cartilage repair matrix in view of the deficiency of animal-derived collagen. The provided recombinant collagen has relatively obvious integrin binding activity, can promote cell adhesion, proliferation and differentiation, and has the effect of repairing cartilage tissue defects.

Integrin binding activity is related to the integrin family, which is a widely distributed transmembrane glycoprotein that is the link between the extracellular matrix and intracellular signal transduction. Integrins are composed of α subunits and β subunits connected by non-covalent bonds to form heterodimers. Currently, 18 α subunits and 8 β subunits have been discovered, which can be combined into at least 24 integrin dimers. The integrin family of cell adhesion molecules is the main connecting substance between the extracellular matrix and parenchymal cells such as inflammatory cells and fibroblasts. It is closely related to cell adhesion, spreading, migration, functional expression, and the occurrence, maintenance and development of tissue fibrosis. It can regulate the interaction between cells and cells and between cells and extracellular matrix, such as recognition and adhesion.

The first object of the present invention is to provide a recombinant collagen, wherein the amino acid sequence of the recombinant collagen comprises N basic repeating units, wherein the basic repeating units contain n1 of the following characteristic amino acid sequences: "G-Xaa ₁ -Xaa ₂ -GE-Xaa ₃ "; the 3' end and the 5' end of the basic repeating unit are connected to form the above characteristic amino acid sequence;

Here, N is an integer greater than or equal to 4; and n1 is an integer greater than or equal to 3.

Furthermore, the N value is an integer of 4 to 300 or more, and further an integer of 4 to 200 or more.

The N value can make the molecular weight of the recombinant collagen reach 1kDa to 250kDa, and further reach 3kDa to 150kDa.

Furthermore, the characteristic amino acid sequences are arranged continuously or at intervals in the basic repeating unit.

Furthermore, the amino acid sequence of the recombinant collagen presents the following characteristics:

[-GE-Xaa ₃ -Yaa ₁ -(G-Xaa ₁ -Xaa ₂ -GE-Xaa ₃ ) _n2 -Yaa ₂ -(G-Xaa ₁ -Xaa ₂ -GE-Xaa ₃ ) _n3 -Yaa ₃ -(G-Xaa ₁ -Xaa ₂ -GE-Xaa ₃ ) _n3 -Yaa ₄ -(G-Xaa ₁ -Xaa ₂ -GE-Xaa ₃ ) _n4 -…………-Yaa _n -(G-Xaa ₁ -Xaa ₂ -GE-Xaa ₃ ) _n -Yaa _n+1 -G-Xaa ₁ -Xaa ₂ -] _N

Wherein, Xaa ₁ is a nonpolar hydrophobic amino acid; Xaa ₂ is one of serine (S), alanine (A), proline (P), and hydroxyproline (O); Xaa ₃ is a basic amino acid;

Each occurrence of Yaa ₁ , Yaa ₂ , Yaa ₃ , Yaa ₄ , ... ... , Yaa _n , Yaa _n+1 is independently selected from none, one or more different or identical amino acids;

n2, n3, n4, ..., n are integers greater than 0, and none of them are 0 at the same time.

The non-polar hydrophobic amino acid of the present invention includes one of glycine (G), valine (V), phenylalanine (F), alanine (A), leucine (L), isoleucine (I), methionine (M), proline (P), and hydroxyproline (O); and further includes one of phenylalanine (F), alanine (A), leucine (L), isoleucine (I), methionine (M), proline (P), and hydroxyproline (O).

The basic amino acid includes one of lysine, arginine and histidine, and is further lysine or arginine.

Furthermore, the recombinant collagen sequence does not contain a protein tag.

In one embodiment of the present invention, the amino acid sequence of the recombinant collagen as mentioned above is shown as SEQID NO.1.

The second object of the present invention is to provide a method for preparing the aforementioned recombinant collagen, which uses gene recombination technology to obtain recombinant collagen through host cell expression, extraction, and purification; the amino acid sequence of the recombinant collagen is obtained by repeatedly connecting multiple basic repeating units;

Furthermore, the amino acid sequence of the recombinant collagen includes N basic repeating units, and the basic repeating unit contains n1 of the following characteristic amino acid sequence: "G-Xaa ₁ -Xaa ₂ -GE-Xaa ₃ "; the 3' end and the 5' end of the basic repeating unit are connected to form the above characteristic amino acid sequence;

Wherein, N is an integer greater than 4; n1 is an integer greater than 3;

Furthermore, the characteristic amino acid sequences are arranged continuously or alternately in the basic repeating unit.

Therefore, the value of N is an integer from 4 to 300, and further an integer from 4 to 200, including but not limited to 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30.......200.

Furthermore, the value of N can make the molecular weight of the recombinant collagen reach 1kDa to 200kDa, and further reach 3kDa to 150kDa.

The value of N can be taken from the endpoint values at both ends of the range value defined by the present invention, or from any integer in the range value. For example, the value of N can be 1, 3, 10, 50, 70, 90, 95, 100, 125, 150, etc.

Further, the recombinant collagen sequence does not contain a protein tag;

The amino acid sequence of the recombinant collagen presents the following characteristics: [-GE-Xaa3-Yaa1-(G-Xaa1-Xaa2-GE-Xaa3)n2-Yaa2-(G-Xaa1-Xaa2-GE-Xaa ₃ ) _n3 -Yaa _3- (G-Xaa ₁ -Xaa ₂ -GE-Xaa ₃ ) _n3 -Yaa _4- (G-Xaa ₁ -Xaa ₂ -GE-Xaa ₃ ) _n4- …………-Yaa _n- (G-Xaa ₁ -Xaa ₂ -GE-Xaa ₃ ) _n -Yaa _n+1 -G-Xaa ₁ -Xaa _2- ] _N

Wherein, Xaa ₁ is a nonpolar hydrophobic amino acid; Xaa ₂ is one of serine (S), alanine (A), proline (P), and hydroxyproline (O); Xaa ₃ is a basic amino acid;

Each occurrence of Yaa ₁ , Yaa ₂ , Yaa ₃ , Yaa ₄ , ... ... , Yaa _n , Yaa _n+1 is independently selected from none, one or more different or identical amino acids;

n2, n3, n4, ..., n are integers greater than 0, and none of them are 0 at the same time.

The third object of the present invention is to provide the use of the aforementioned recombinant collagen in the preparation of products for regulating cell adhesion, proliferation and differentiation.

The fourth object of the present invention is to provide the use of the aforementioned recombinant collagen in preparing a cartilage repair matrix product.

When the recombinant collagen of the present invention is used in the aforementioned purposes, it can be used in combination with polymers and/or their derivatives, wherein the polymers include natural polymers and synthetic polymers; further, the natural polymers include but are not limited to hyaluronic acid, chitosan, alginate, cellulose, etc.; further, the synthetic polymers include but are not limited to PLA, PGA, PLCL, PVA.

When the recombinant collagen of the present invention is used in the aforementioned purposes, the concentration used is 0.01 to 100 mg/mL, for example, it can be 0.01 mg/mL, 0.05 mg/mL, 2 mg/mL, 4.5 mg/mL, 7 mg/mL, 10 mg/mL, 15 mg/mL, 20 mg/mL, 50 mg/mL, 80 mg/mL, 100 mg/mL, etc. The concentration used is related to the application site and scenario.

The fifth object of the present invention is to provide a cartilage repair matrix, comprising a polymer and/or its derivatives, and the aforementioned recombinant collagen.

Furthermore, the polymer includes natural polymers and synthetic polymers; further, the natural polymer is at least selected from any one of hyaluronic acid, chitosan, alginate, and cellulose; further, the synthetic polymer is at least selected from any one of PLA, PGA, PLCL, and PVA.

The sixth object of the present invention is to provide a method for preparing the aforementioned cartilage repair matrix, comprising the following steps:

(1) dissolving the hyaluronic acid derivative in a buffer solution and adding EDC/sNHS solution for reaction;

(2) adding the recombinant collagen to the reaction system in step (1) to react, dialyzing and drying the solution obtained after the reaction to obtain a composite matrix;

(3) After the composite matrix is dissolved, a photoinitiator solution is added to obtain a cartilage repair matrix;

Humanized collagen has a small molecular weight and poor mechanical properties. It is difficult to form a gel after modification and cannot meet the needs of cartilage tissue engineering. In the embodiment of the present invention, hyaluronic acid is firstly appropriately chemically modified with methacrylic anhydride to introduce unsaturated carbon-carbon double bonds, and then the carboxyl groups in the hyaluronic acid are activated by adding EDC/sNHS, and then recombinant humanized collagen is added to make the amino groups on the protein and the activated carboxyl groups on the hyaluronic acid undergo amidation reaction to couple the two. The composite hydrogel finally prepared by photocrosslinking not only has good mechanical properties and biocompatibility, but also the photocrosslinking method makes the operation easier, which can meet the needs of cartilage tissue engineering, and also meet the filling of irregular defects in clinical practice.

The EDC described in the present invention refers to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and NHS refers to N-hydroxysuccinimide.

Furthermore, the polymer derivative in step (1) is hyaluronic acid modified with methacrylic anhydride;

Further, the molecular weight of the modified hyaluronic acid is 100 to 1500 kDa, preferably 300 to 600 kDa, more preferably 300 kDa;

Furthermore, the degree of grafting of the hyaluronic acid modification in step (1) is 20 to 60%, preferably 30 to 50%;

Furthermore, in step (1), after EDC and sNHS are added, the concentration of sNHS is 0.5-5 mM, and the concentration of EDC is 0.5-5 mM; further, the concentration of sNHS is 1-3 mM, and the concentration of EDC is 2-4 mM.

In the present invention, in step (1), the pH value of the mixed solution after adding the EDC/sNHS solution is 4 to 5, preferably 4.75;

The pH value of the mixed solution after the reaction is 7-8, and the reaction time is 2-5 hours; preferably, the pH value is 7.4, and the reaction time is 3 hours.

In the present invention, in step (2), the mass ratio of recombinant collagen to polymer derivative is 0.5-30:2-10; further 0.5-20:6; further 1-11:6;

Furthermore, the reaction time in step (2) is 5 to 15 hours, preferably 8 to 12 hours;

Furthermore, in step (2), the solution after the reaction is placed in a dialysis bag with a molecular weight cutoff of 8000 to 14000, dialyzed with deionized water for 2 to 4 days, and then freeze-dried to obtain a composite matrix.

The photoinitiator used in the present invention is at least one of LAP and 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (I2959), and the final concentration is 0.01-0.05% by mass volume, preferably 0.05%.

In the present invention, the concentration of the composite matrix after adding the photoinitiator in step (3) is 2 to 30 mg/ml, further 5 to 20 mg/ml, and further 7 to 17 mg/ml.

The cartilage repair matrix of the present invention is a product containing the recombinant collagen, which can be used as a component of medical devices including but not limited to implant materials, tissue engineering scaffold materials, soft tissue filling materials, cells or other active substance carrier materials.

When used clinically, the cartilage repair matrix of the present invention can be prepared into a cartilage repair hydrogel after adding cells, or can be directly applied to the cartilage defect site without adding cells.

The seventh object of the present invention is to provide a method for preparing a cartilage repair hydrogel, wherein the cartilage repair matrix prepared by the aforementioned method is compounded with chondrocytes or bone marrow mesenchymal stem cells, injected into the cartilage damage site, and irradiated with light to form a cartilage repair hydrogel.

The wavelength of the light is selected adaptively according to the initiator used.

The cartilage repair hydrogel of the present invention further comprises a physiologically acceptable carrier material.

The carrier materials here include but are not limited to water-soluble carrier materials (such as polyethylene glycol, polyvinyl pyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.).

The composition can also be used in combination with other functional materials as needed, such as components with antibacterial and antimicrobial effects, anti-aging components, anticoagulant components, antioxidant components, growth factors, etc.

The composition can be prepared according to conventional techniques of pharmaceutics or cosmetics, including mixing the recombinant collagen of the present invention as an active ingredient with a carrier, and preparing the desired dosage form according to conventional techniques. The composition of the present invention can be formulated into a variety of dosage forms as required, including but not limited to oral administration preparations, mucosal administration preparations, injection preparations, inhalation preparations, and external preparations.

The products described in the present invention include but are not limited to medicines, foods, health products or cosmetics, and daily necessities.

In this specification, the word "may" includes both performing a certain process and not performing a certain process.

In practical applications, the recombinant collagen of the present invention and the above-mentioned composition can be directly administered to patients as drugs, or can be administered to patients after being mixed with suitable carriers or excipients. The carrier materials here include but are not limited to water-soluble carrier materials (such as polyethylene glycol, polyvinyl pyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Among them, water-soluble carrier materials are preferred. These materials can be made into a variety of dosage forms, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, freeze-dried powder injections, etc. Among them, the suppository can be a vaginal suppository, a vaginal ring, or an ointment, cream or gel suitable for vaginal application.

The dosage form can be a common preparation, a sustained-release preparation, a controlled-release preparation and various microparticle delivery systems. In order to prepare the unit dosage form into tablets, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and binders, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, acacia paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methylcellulose, potassium phosphate, polyvinyl pyrrolidone, etc.; disintegrants, such as dry starch, alginate, agar powder, brown seaweed starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid esters, sodium lauryl sulfate, methylcellulose, ethylcellulose, etc.; disintegration inhibitors, such as sucrose, tristearin, cocoa butter, hydrogenated oil, etc.; absorption promoters, such as quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants, such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. Tablets can also be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets. In order to make unit dosage forms into pills, various carriers known in the art can be widely used. Examples of carriers are, for example, diluents and absorbents, such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinyl pyrrolidone, Gelucire, kaolin, talc, etc.; adhesives such as gum arabic, tragacanth gum, gelatin, ethanol, honey, liquid sugar, rice paste or flour paste, etc.; disintegrants, such as agar powder, dry starch, alginate, sodium dodecyl sulfate, methylcellulose, ethylcellulose, etc. In order to make unit dosage forms into suppositories, various carriers known in the art can be widely used. Examples of carriers are, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, etc. In order to prepare the unit dosage form into an injection preparation, such as a solution, emulsion, lyophilized powder injection and suspension, all diluents commonly used in the art can be used, for example, water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxygenated isostearyl alcohol, polyoxyethylene sorbitol fatty acid esters, etc. In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerol can be added to the injection preparation. In addition, conventional cosolvents, buffers, pH adjusters, etc. can also be added. In addition, if necessary, colorants, preservatives, fragrances, flavoring agents, sweeteners or other materials can also be added to the pharmaceutical preparation.

The above dosage forms can be administered by injection, including subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, intracisternal injection or infusion, etc.; cavity administration, such as rectal, vaginal and sublingual; respiratory tract administration, such as nasal cavity; mucosal administration.

The above-mentioned administration route is preferably injection, and the preferred injection route is subcutaneous injection.

The dosage of the recombinant collagen of the present invention and the above-mentioned composition depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight and individual response of the patient or animal, the specific active ingredient used, the route of administration and the number of administrations, etc. The above-mentioned dosage can be administered in a single dosage form or divided into several, such as two, three or four dosage forms. For any specific patient, the specific therapeutically effective dosage level must be determined based on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific active ingredient used; the specific composition used; the patient's age, weight, general health, sex and diet; the administration time, route of administration and excretion rate of the specific active ingredient used; the duration of treatment; drugs used in combination or simultaneously with the specific active ingredient used; and similar factors known in the medical field.

For example, it is common practice in the art to start doses of the active ingredient at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

Beneficial effects:

(1) The recombinant collagen provided by the present invention has relatively obvious integrin binding activity, has the effect of promoting cell adhesion, proliferation, differentiation, and repairing cartilage tissue defects, and has good application prospects.

(2) The present invention covalently cross-links hyaluronic acid and humanized collagen to form a stable composite hydrogel matrix with good mechanical properties and can well maintain the morphology of the composite; it has good biocompatibility, and the photocross-linking method makes the operation easier, which can meet the needs of cartilage tissue engineering and also meet the clinical filling of irregular defects.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the results of fibroblast cell adhesion and growth;

Figure 2 shows the OD values of fibroblast cell adhesion;

Figure 3 shows the results of chondrocyte adhesion and growth;

Figure 4 shows the OD values of chondrocyte adhesion;

FIG5 is the results of the adhesion experiment of sequence A and sequence B at different concentrations to human gingival fibroblasts;

FIG6 is a morphological diagram of chondrocyte/hydrogel complexes after 1, 7, 14, and 21 days of in vitro culture;

FIG7 shows the OD values of chondrocyte adhesion after 1, 7, and 14 days of in vitro culture of chondrocyte/hydrogel complexes;

FIG8 is a CCK-8 graph of chondrocytes after 1, 7, and 14 days of in vitro culture of chondrocyte/hydrogel complexes;

FIG9 is the staining result of chondrocyte/hydrogel complex;

FIG10 is the result of immunohistochemical staining of type II collagen of chondrocyte/hydrogel complex;

FIG. 11 shows the quantitative detection results of GAG in chondrocyte/hydrogel complex.

Detailed ways

The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

Example 1

A nucleotide sequence encoding the recombinant collagen is constructed, a vector containing the nucleotide sequence is constructed and screened, a host cell (which may be a prokaryotic cell or a eukaryotic cell) containing the vector is constructed and screened, the host cell is cultured under appropriate conditions to express the protein, and the expressed recombinant collagen is collected and purified.

As described in the present invention, the term "vector" is a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes (PAC) derived from P1; bacteriophages such as lambda phage or M13 phage and animal viruses. The vector may contain a variety of elements for controlling expression, including but not limited to promoter sequences, transcription start sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site. The vector may contain the nucleic acid of the present invention to facilitate introduction into cells for expression. The vector may contain expression control elements operably linked to the nucleic acid, such as promoters, terminators and/or enhancers.

The gene recombination and transcription involved in the present invention refers to synthesizing a nucleotide sequence encoding recombinant collagen, cloning the nucleotide sequence into an expression vector by conventional techniques, and then transforming the expression vector into a host cell to construct and screen an engineered strain. The transformation method includes but is not limited to electroporation, _CaCl2 transformation, etc.; the expression vector can be a commonly used vector such as pET26, pET32, pGEX-6p, pPIC9, pPIC9K, etc., and can be a plasmid, phage, virus or other vector.

The term "host cell" as used herein refers to a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. These techniques include transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration. The host cell can be a eukaryotic cell or a prokaryotic cell. For example, microbial cells such as Enterobacteriaceae cells (e.g., Escherichia coli BL21, DH5α, etc.), eukaryotic cells such as yeast (e.g., Pichia pastoris, Saccharomyces cerevisiae, etc.) or CHO cells.

The fermentation culture involved in the present invention refers to inducing fermentation culture in a sterile fermentation tank by combining the screened and protein-high expression-identified engineered strain with a suitable fermentation culture medium under suitable temperature, pressure and pH conditions.

The separation and purification of proteins involved in the present invention refers to obtaining bacterial liquid after lysing, homogenizing, separating and other treatments of bacteria, and further obtaining recombinant collagen by conventional protein separation and purification techniques. Separation and purification techniques include but are not limited to filtration, centrifugation, salting out, dialysis, liquid chromatography, ion exchange chromatography, affinity chromatography, etc.

In the embodiment of the present invention, gene recombination and transcription are performed by taking Escherichia coli as a host cell, specifically:

1. Preparation of recombinant collagen by E. coli fermentation

Gene recombination and transcription: The DNA fragments were codon optimized and spliced and recombined using the PCR method. The expression vector was constructed using pET-32a and then transferred into the Escherichia coli expression strain BL21. After culture and screening, genetically engineered Escherichia coli bacteria with high protein expression were obtained.

Fermentation culture: Select a single colony of genetically engineered Escherichia coli from the LB plate, place it in a 100mL Erlenmeyer flask containing 10mL LB medium, and culture it at 37℃, 220rpm for 12-16h; inoculate the bacterial solution into a fermenter containing LB medium at a ratio of 1:100 for scale-up culture, culture it at 37℃, 220rpm for 3h until OD600 is about 0.6, add 0.5mM IPTG, induce culture at 16℃ for 20h, and then collect the bacteria by centrifugation.

Protein separation and purification: After reselecting the bacteria with Tris buffer, stir at high speed to completely dissolve the bacteria, collect the supernatant by centrifugation and cool to 4°C; the supernatant is filtered using 1μm, 0.45μm and 0.22μm filter membranes in sequence and further purified by affinity chromatography to obtain recombinant collagen.

2. Cell culture method

1) Sample preparation: Recombinant collagen was prepared into a certain concentration solution (such as 0.5 mg/ml) with PBS, and the animal source collagen solution was diluted to the same concentration (such as 0.5 mg/ml) with PBS, and PBS was used as the blank control group;

2) Plating: Add the experimental solution to each well of a 96-well plate, add 100 μL to each well, 5 replicates per group, and incubate at 4°C overnight;

3) Blocking: After the plating is completed, remove the liquid in the well plate and wash it twice with 200 μL PBS solution, add 100 μL of heat-inactivated 1% BSA-PBS solution, and incubate in an incubator at 37°C and 5% _CO2 for 1 hour;

4) Cell seeding: After the incubation, remove the liquid in the well plate and wash twice with 200 μL PBS solution, add 100 μL of cell suspension with a density of 5×10 ⁴ -1×10 ⁶ cells/ml to each well, and incubate in an incubator at 37°C and 5% CO ₂ for 1 hour;

5) Detection: After the incubation, remove the liquid in the well plate and wash twice with 200 μL PBS solution, add 150 μL CCK-8, incubate in an incubator at 37°C, 5% _CO2 for 1 hour, then draw 100 μL of the detection solution and add it to a new 96-well plate to detect the absorbance value (OD) at 450 nm.

Test Example 1:

The method of Example 1 was used to prepare the following three recombinant collagens containing different amino acid sequences.

The recombinant collagen obtained by directly linking the repeating units with the above multiple characteristic amino acid sequence combinations for 16 times was used to improve the adhesion, migration, functional expression and other experiments of cells. Sequence A was linked 16 times, sequence A: GER GAP GFR GPA GPN GIP GEK GPA GER GAP, and its full sequence is as follows (SEQ ID NO.1):

Another recombinant collagen protein obtained by directly repeating the repeating unit with different characteristic amino acid sequences has different effects on cell adhesion and proliferation. Repeat the connecting sequence B 4 times, sequence B: GEK GSP GAD GPA GAP GTP GPQ GIA GQR GVV GLP GQR GER GFP GLP GPS GEP GKQ GPS GAS, and its full sequence is as follows (SEQ ID NO.2):

After the recombinant collagen with the above repeating unit connected 16 times was added with a tag, the adhesion and proliferation effects on cells were significantly reduced; the sequence C was repeatedly connected 16 times, sequence C: HHHHHH GER GAP GFR GPA GPN GIP GEK GPA GER GAP, and its full sequence is as follows (SEQ ID NO.3):

The cell culture method in Example 1 was used to perform fibroblast adhesion test, chondrocyte adhesion test, and human gingival fibroblast adhesion promotion test on the above three collagens.

The experimental results are shown in Figures 1 to 3.

The results in Figure 1 show that the recombinant collagen experimental group obtained by repeatedly connecting sequence A with more characteristic amino acid sequence combinations had very obvious fibroblast adhesion and good cell morphology; the recombinant collagen experimental group obtained by repeatedly connecting sequence B after reducing the characteristic amino acid sequence had similar fibroblast adhesion and cell morphology as the animal collagen group; and the fibroblast adhesion of the recombinant collagen group obtained by repeatedly connecting sequence C after adding a label was significantly reduced, and there was very little fibroblast adhesion in the blank control group.

The results in Figure 2 show that the recombinant collagen experimental group obtained by repeatedly connecting sequence A with more characteristic amino acid sequence combinations can best promote the adhesion of fibroblasts, followed by animal collagen and recombinant collagen obtained by repeatedly connecting sequence B. The adhesion amount of fibroblasts in these three groups is significantly higher than that of the recombinant collagen obtained by repeatedly connecting sequence C and the blank control; the adhesion amount of fibroblasts in the sequence C recombinant collagen group is slightly higher than that of the blank control.

The results in Figure 3 show that the adhesion of chondrocytes in the animal collagen group was the most obvious, almost covering the entire field of view; followed by the sequence A recombinant collagen group, where the chondrocyte adhesion was obvious and relatively concentrated, with good cell morphology; the adhered chondrocytes in the sequence B recombinant collagen group were relatively dispersed, with only a small number concentrated, and good cell morphology; the adhered chondrocytes in the sequence C recombinant collagen group were dispersed and the number was small; in the blank control group, only a small number of chondrocytes adhered and were dispersed.

The results in Figure 4 show that animal collagen is most conducive to promoting the adhesion of chondrocytes, which is significantly higher than other groups; the sequence A recombinant collagen group with more characteristic amino acid sequence combinations is significantly higher than the sequence B recombinant collagen group, sequence C recombinant collagen group and blank control group; the sequence B recombinant collagen group is significantly higher than the sequence C recombinant collagen group and blank control group; the sequence C recombinant collagen group is slightly higher than the blank control group, but there is no statistical difference.

Figure 5 The results of the human gingival fibroblast adhesion promotion experiment show that the sequence A recombinant collagen group with more characteristic amino acid sequence combinations is more conducive to the adhesion of human gingival fibroblasts than sequence B at different concentrations; as the concentration of recombinant collagen increases, the adhesion of human gingival fibroblasts also gradually increases.

Example 2 Application of recombinant collagen in promoting cartilage repair

[Corrected 13.12.2023 in accordance with Article 91]
The method of Example 1 is used to prepare recombinant collagen that meets the amino acid sequence characteristics of the present invention, wherein the amino acid sequence core sequence of the recombinant humanized collagen is preferably GERGAPGFRGPAGPNGIPGEKGPAGERGAP (sequence A), which is repeatedly connected 16 times and used to promote cartilage repair. The specific steps are:

(1) dissolving methacrylated hyaluronic acid (HA-MA) with a molecular weight of 300 KDa and a modification degree of 37% in a MES buffer solution to obtain a 1 mM methacrylated hyaluronic acid solution;

(2) solid sNHS and solid EDC are sequentially added to the mixed solution to make the concentration of sNHS 2 mM and the concentration of EDC 3 mM, and the pH of the mixed solution is maintained at 4.75 with 1.0 M NaOH solution or 1.0 M HCl solution;

(3) Stirring the reaction at room temperature for 2 h;

(4) Adjust the pH of the reaction system to 7.4 with 1.0 M NaOH solution;

(5) adding recombinant humanized collagen to the reaction system so that the mass ratio of recombinant humanized collagen to methacrylated hyaluronic acid in the mixed solution is 1:6, and stirring the reaction for 8 hours;

(6) The reaction solution was placed in a dialysis bag with a molecular weight cutoff of 8000-14000, dialyzed with deionized water for three days, and then freeze-dried using a freeze dryer to obtain freeze-dried sponge HA-rhCol III;

(7) Dissolve the freeze-dried sponge in 0.01 M PBS and add LAP photoinitiator solution (final concentration: 0.05% by mass volume) to make the final concentration of HA-rhCol III solution 7 mg/ml;

(8) Take the precursor solution in step (7) and place it in a silicone mold with a specification of Ф6mm×2.5mm. Irradiate it under ultraviolet light for 1 minute to obtain hyaluronic acid hydrogel containing recombinant humanized collagen (HA-rhCol III).

Example 3 Application of recombinant collagen in promoting cartilage repair

[Corrected 13.12.2023 in accordance with Article 91]
The method of Example 1 is used to prepare recombinant collagen that meets the amino acid sequence characteristics of the present invention, wherein the amino acid sequence core sequence of the recombinant humanized collagen is preferably GERGAPGFRGPAGPNGIPGEKGPAGERGAP (sequence A), which is repeatedly connected 16 times and used to promote cartilage repair. The specific steps are:

(1) dissolving methacrylated hyaluronic acid HA-MA with a molecular weight of 100 KDa and a modification degree of 50% in a MES buffer solution to obtain a 2 mM methacrylated hyaluronic acid solution;

(2) solid sNHS and solid EDC are sequentially added to the mixed solution to make the concentration of sNHS 3 mM and the concentration of EDC 5 mM, and the pH of the mixed solution is maintained at 4.75 with 0.5 M NaOH solution or 0.5 M HCl solution;

(3) Stirring the reaction at room temperature for 2 h;

(4) Adjust the pH of the reaction system to 7.4 with 1.0 M NaOH solution;

(5) adding recombinant humanized collagen to the reaction system so that the mass ratio of recombinant humanized collagen to methacrylated hyaluronic acid in the mixed solution is 11:6, and stirring the reaction for 12 hours;

(6) The reaction solution was placed in a dialysis bag with a molecular weight cutoff of 8000-14000, dialyzed with deionized water for three days, and then freeze-dried using a freeze dryer to obtain the product HA-rhCol III;

(7) Dissolve the freeze-dried sponge in 0.01 M PBS and add LAP photoinitiator solution (final concentration: 0.01% by mass volume) to make the final concentration of HA-rhCol III solution 17 mg/ml;

(8) Take the precursor solution in step (7) and place it in a silicone mold with a specification of Ф6mm×2.5mm. Irradiate it under ultraviolet light for 1 minute to obtain HA-rhCol III hydrogel.

Example 4 Application of recombinant collagen in promoting cartilage repair

[Corrected 13.12.2023 in accordance with Article 91]
The method of Example 1 is used to prepare recombinant collagen that meets the amino acid sequence characteristics of the present invention, wherein the amino acid sequence core sequence of the recombinant humanized collagen is preferably GERGAPGFRGPAGPNGIPGEKGPAGERGAP (sequence A), which is repeatedly connected 16 times (SEQ ID NO. 1), and is used to promote cartilage repair. The specific steps are:

(1) dissolving methacrylated hyaluronic acid HA-MA with a molecular weight of 600 KDa and a modification degree of 30% in MES buffer solution to obtain a 1 mM methacrylated hyaluronic acid solution;

(2) sNHS and EDC were added to the mixed solution in sequence, so that the concentration of sNHS was 1 mM and the concentration of EDC was 3 mM, and the pH of the mixed solution was maintained at 4.75 with 1.0 M NaOH solution or 1.0 M HCl solution;

(3) Stirring the reaction at room temperature for 3 h;

(4) Adjust the pH of the reaction system to 7.4 with 1.0 M NaOH solution;

(5) adding recombinant humanized collagen to the reaction system so that the mass ratio of recombinant humanized collagen to methacrylated hyaluronic acid in the mixed solution is 0.5:6, and stirring the reaction for 8 hours;

(6) The reaction solution was placed in a dialysis bag with a molecular weight cutoff of 8000-14000, dialyzed with deionized water for three days, and then freeze-dried using a freeze dryer to obtain the product HA-rhCol III;

(7) Dissolve the freeze-dried sponge in 0.01 M PBS and add LAP photoinitiator solution (final concentration: 0.05% by mass volume) to make the final concentration of HA-rhCol III solution 7 mg/ml;

(8) Take the precursor solution in step (7) and place it in a silicone mold with a specification of Ф6mm×2.5mm. Irradiate it under ultraviolet light for 2 minutes to obtain HA-rhCol III hydrogel.

Example 5 Application of recombinant collagen in promoting cartilage repair

[Corrected 13.12.2023 in accordance with Article 91]
The method of Example 1 is used to prepare recombinant collagen that meets the amino acid sequence characteristics of the present invention, wherein the amino acid sequence core sequence of the recombinant humanized collagen is preferably GERGAPGFRGPAGPNGIPGEKGPAGERGAP, which is repeated 16 times (SEQ ID NO. 1), and is used to promote cartilage repair. The specific steps are:

(1) dissolving methacrylated hyaluronic acid HA-MA with a molecular weight of 1000 KDa and a modification degree of 40% in a MES buffer solution to obtain a 0.6 mM methacrylated hyaluronic acid solution;

(2) sNHS and EDC were added to the mixed solution in sequence to make the concentration of sNHS 5 mM and the concentration of EDC 5 mM, and the pH of the mixed solution was maintained at 4.75 with 0.2 M NaOH solution or 0.2 M HCl solution;

(3) Stirring the reaction at room temperature for 3 h;

(4) Adjust the pH of the reaction system to 7.4 with 0.5 M NaOH solution;

(5) adding recombinant humanized collagen to the reaction system so that the mass ratio of recombinant humanized collagen to methacrylated hyaluronic acid in the mixed solution is 11:6, and stirring the reaction for 12 hours;

(6) The reaction solution was placed in a dialysis bag with a molecular weight cutoff of 8000-14000, dialyzed with deionized water for three days, and then freeze-dried using a freeze dryer to obtain the product HA-rhCol III;

(7) Dissolve the freeze-dried sponge in 0.01 M PBS and add I2959 photoinitiator solution (final concentration: 0.03% by mass volume) to make the final concentration of HA-rhCol III solution 17 mg/ml;

(8) Take the precursor solution in step (7) and place it in a silicone mold with a specification of Ф6mm×2.5mm. Irradiate it under ultraviolet light for 1 minute to obtain HA-rhCol III hydrogel.

Test Example 2:

The results of the cell experiment of the recombinant humanized collagen modified hyaluronic acid hydrogel prepared in Example 2 are as follows:

Combined with the results of Figures 6 and 7, the chondrocytes in the HA-MA hydrogel were cultured for 21 days. Due to the lack of adhesion sites, the chondrocytes aggregated and grew in situ. However, the chondrocytes in the HA-rhCol III hydrogel were partially spread out at 7 days. At 14 and 21 days, the cell spreading growth phenomenon was more obvious and evenly distributed. This shows that rhCol III is conducive to cell adhesion, migration and proliferation, and the HA-rhCol III hydrogel can promote the proliferation of chondrocytes better than the HA-MA hydrogel.

The results in Figure 8 show that the nuclei of cells in the HA-rhCol III hydrogel group did not show deformation or shrinkage, and at 7 and 14 days of culture, the actin of cells in the two groups of hydrogels was spindle-shaped, indicating that the cells were well spread inside the gel, while the cells in the HA-MA group proliferated in situ in the form of cell clusters at each time point. This result shows that rhCol III provides a suitable site for cell adhesion, allowing chondrocytes to adhere faster and providing conditions for cell proliferation.

The results of histological and type II collagen immunohistochemical staining (Figures 9-10) showed that there were more obvious polysaccharide and protein staining areas in the HA-rhCol III gel, indicating that the addition of rhCol III can promote the functional expression of chondrocytes. The results of GAG quantitative detection (Figure 11) further confirmed that the addition of rhCol III is more conducive to promoting the secretion of extracellular matrix of chondrocytes.

The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims (19)

A recombinant collagen, characterized in that the amino acid sequence of the recombinant collagen comprises N basic repeating units, wherein the basic repeating units contain n1 of the following characteristic amino acid sequences: "G-Xaa ₁ -Xaa ₂ -GE-Xaa ₃ "; the 3' end and the 5' end of the basic repeating unit are connected to form the above characteristic amino acid sequence; Here, N is an integer greater than or equal to 4; and n1 is an integer greater than or equal to 3. The recombinant collagen according to claim 1, wherein N is an integer from 4 to 300. The recombinant collagen according to claim 1, wherein N is an integer from 4 to 200. The recombinant collagen according to claim 1, characterized in that the characteristic amino acid sequence is arranged continuously or at intervals in the basic repeating unit. The recombinant collagen according to claim 1, characterized in that the amino acid sequence of the recombinant collagen presents the following characteristics:
[-GE-Xaa ₃ -Yaa ₁ -(G-Xaa ₁ -Xaa ₂ -GE-Xaa ₃ ) _n2 -Yaa ₂ -(G-Xaa ₁ -Xaa ₂ -GE-Xaa ₃ ) _n3 -
Yaa ₃ -(G-Xaa ₁ -Xaa ₂ -GE-Xaa ₃ ) _n3 -Yaa ₄ -(G-Xaa ₁ -Xaa ₂ -GE-Xaa ₃ ) _n4 -…………-Yaa _n -(G-Xaa ₁ -Xaa ₂ -GE-Xaa ₃ ) _n -Yaa _n+1 -G-Xaa ₁ -Xaa ₂ -] _N Wherein, Xaa ₁ is a nonpolar hydrophobic amino acid; Xaa ₂ is one of serine (S), alanine (A), proline (P), and hydroxyproline (O); Xaa ₃ is a basic amino acid; Each occurrence of Yaa ₁ , Yaa ₂ , Yaa ₃ , Yaa ₄ , ... ... , Yaa _n , Yaa _n+1 is independently selected from none, one or more different or identical amino acids; n2, n3, n4, ..., n are integers greater than 0, and none of them are 0 at the same time. The recombinant collagen according to claim 1, wherein the recombinant collagen sequence does not contain a protein tag. The recombinant collagen as described in any one of claims 1 to 6 is characterized in that the amino acid sequence of the recombinant collagen is as shown in SEQID NO.1. The method for preparing the recombinant collagen according to any one of claims 1 to 7 is characterized in that the recombinant collagen is obtained by expressing, extracting and purifying the recombinant collagen through host cells using gene recombination technology; the amino acid sequence of the recombinant collagen is obtained by repeatedly connecting multiple basic repeating units. The method for preparing recombinant collagen according to claim 8, characterized in that the amino acid sequence of the recombinant collagen comprises N basic repeating units, wherein the basic repeating units contain n1 of the following characteristic amino acid sequences: "G-Xaa ₁ -Xaa ₂ -GE-Xaa ₃ "; the 3' end and the 5' end of the basic repeating unit are connected to form the above characteristic amino acid sequence; Here, N is an integer greater than or equal to 4; and n1 is an integer greater than or equal to 3. The method for preparing recombinant collagen as claimed in claim 9, characterized in that the characteristic amino acid sequence is arranged continuously or at intervals in the basic repeating unit. The method for preparing recombinant collagen according to claim 8, wherein the recombinant collagen sequence does not contain a protein tag; The amino acid sequence of the recombinant collagen presents the following characteristics:
[-GE-Xaa ₃ -Yaa ₁ -(G-Xaa ₁ -Xaa ₂ -GE-Xaa ₃ ) _n2 -Yaa ₂ -(G-Xaa ₁ -Xaa ₂ -GE-Xaa ₃ ) _n3 -
Yaa ₃ -(G-Xaa ₁ -Xaa ₂ -GE-Xaa ₃ ) _n3 -Yaa ₄ -(G-Xaa ₁ -Xaa ₂ -GE-Xaa ₃ ) _n4 -…………-Yaa _n -(G-Xaa ₁ -Xaa ₂ -GE-Xaa ₃ ) _n -Yaa _n+1 -G-Xaa ₁ -Xaa ₂ -] _N Wherein, Xaa ₁ is a nonpolar hydrophobic amino acid; Xaa ₂ is one of serine (S), alanine (A), proline (P), and hydroxyproline (O); Xaa ₃ is a basic amino acid; Each occurrence of Yaa ₁ , Yaa ₂ , Yaa ₃ , Yaa ₄ , ... ... , Yaa _n , Yaa _n+1 is independently selected from none, one or more different or identical amino acids; n2, n3, n4, ..., n are integers greater than 0, and none of them are 0 at the same time. Use of the recombinant collagen according to any one of claims 1 to 7 in the preparation of a product for regulating cell adhesion, proliferation and differentiation. Use of the recombinant collagen according to any one of claims 1 to 7 in the preparation of a cartilage repair matrix product. A cartilage repair matrix, characterized in that it comprises a polymer and/or its derivatives, and the recombinant collagen according to any one of claims 1 to 7; the polymer comprises a natural polymer and a synthetic polymer. The cartilage repair matrix according to claim 14, characterized in that the natural polymer is at least selected from any one of hyaluronic acid, chitosan, alginate, and cellulose; and the synthetic polymer is at least selected from any one of PLA, PGA, PLCL, and PVA. The method for preparing the cartilage repair matrix according to claim 15, characterized in that it comprises the following steps: (1) Dissolving the polymer derivative in a buffer solution and adding EDC/sNHS solution for reaction; (2) adding the recombinant collagen according to any one of claims 1 to 7 to the reaction system in step (1) to react, dialyzing and drying the solution obtained after the reaction to obtain a composite matrix; (3) After the composite matrix is dissolved, a photoinitiator solution is added to obtain a cartilage repair matrix. The method for preparing a cartilage repair matrix according to claim 16, characterized in that the polymer derivative described in step (1) is hyaluronic acid modified with methacrylic anhydride; the molecular weight of the modified hyaluronic acid is 100 to 1500 kDa; the degree of grafting of the modified hyaluronic acid is 20 to 60%; the pH value of the mixed solution after adding the EDC/sNHS solution is 4 to 5; after adding EDC and sNHS, the concentration of sNHS is 0.5 to 5 mM, and the concentration of EDC is 0.5 to 5 mM; the pH value of the mixed solution after the reaction is 7 to 8, and the reaction time is 2 to 5 hours; In step (2), the mass ratio of recombinant collagen to polymer derivative is 0.5-30:2-10; the reaction time is 5-15 hours; the solution after the reaction is placed in a dialysis bag with a molecular weight cutoff of 8000-14000, dialyzed with deionized water for 2-4 days, and then freeze-dried to obtain a composite matrix; The concentration of the composite matrix after adding the photoinitiator in step (3) is 2 to 30 mg/ml. The method for preparing a cartilage repair matrix according to claim 17, characterized in that, in step (1), the molecular weight of the modified hyaluronic acid is 300 to 600 kDa; the degree of grafting of the modified hyaluronic acid is 30 to 50%; In step (2), the mass ratio of recombinant collagen to polymer derivative is 0.5 to 20:6, and the reaction time is 8 to 12 hours; The concentration of the composite matrix after adding the photoinitiator in step (3) is 5 to 20 mg/ml. A method for preparing a cartilage repair hydrogel, characterized in that the cartilage repair matrix prepared by the method according to any one of claims 16 to 18 is compounded with chondrocytes or bone marrow mesenchymal stem cells, injected into a cartilage injury site, and irradiated with light to form a cartilage repair hydrogel.